The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined sub-nanometer resolution cryo-electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.The protein-conducting channel (PCC) of the canonical secretory pathway is formed in all cells by the Sec61/SecY complex. It engages in the post-and co-translational translocation of secretory proteins across, and the insertion of integral membrane proteins into the membrane of the endoplasmic reticulum (ER) in eukaryotes and the plasma membrane of bacteria (1,2).# To whom correspondence should be addressed. beckmann@lmb.uni-muenchen.de; elisabet.mandon@umassmed.edu. In the co-translational translocation mode the ribosome with an emerging signal sequence is targeted to the membrane by the signal recognition particle (SRP) and its receptor (3). Here, the Sec complex acts as a receptor for the ribosome via its cytosolic loops (4). The alignment of the ribosomal tunnel with a central pore of the PCC allows direct movement of the nascent chain from the ribosomal tunnel exit across or into the membrane (5,6).The PCC-forming heterotrimeric Sec complex consists of one large subunit (Sec61α in Mammalia, Sec61p/Ssh1p in yeast, SecY in Bacteria) and two small subunits (Sec61β, γ in eukaryotes and SecE, G in Bacteria). Conflicting models have been presented as to how many of these heterotrimers are necessary to build an active PCC and what the actual path of the polypeptide chain is. The Escherichia coli SecYEG complex forms back-to-back dimers in two-dimensional crystals (7), and low resolution single particle electron microscopic (EM) data revealed a pentagonal ringlike morphology of the PCC interpreted as oligomers (5,6,(8)(9)(10)(11)(12). The monomeric crystal structure of an archaeal SecYEß complex (13), in combination with chemical cross-linking data (14), led to the interpretation that a single copy of the Sec complex is sufficient to serve as an active PCC, even when assembled into a dimer for posttranslational translocation (15) Cryo-EM and 3D reconstructionFor structure determination by cryo-EM we used digitonin-solubilized purified Ssh1 complex (Sec sixty-one homologue 1 from the yeast Saccharomyces cerevisiae) containing Ssh1p, Sbh2p and Sss1p (19). This complex is active in the co-translational translocation mode only, i.e. when ribosome-bound (20,21).We reconstituted the Ssh1 complex with in vitro progra...

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.modeling | molecular dynamics | flexible fitting I n all living cells, the translation of mRNA into polypeptide occurs on ribosomes. Ribosomes provide a platform upon which aminoacyl-tRNAs interact with the mRNA as well as position the aminoacyl-tRNAs for peptide-bond formation (1). Ribosomes are composed of two subunits, a small subunit that monitors the mRNA-tRNA codon-anticodon duplex to ensure fidelity of decoding (2, 3) and a large subunit that contains the active site where peptide-bond formation occurs (4). Both the small and large subunits are composed of RNA and protein: In eubacteria such as Escherichia coli, the small subunit contains one 16S rRNA and 21 ribosomal proteins (r proteins), whereas the large subunit contains 5S and 23S rRNAs and 33 r proteins. Crystal structures of the complete bacterial 70S ribosome were initially reported at 5.5 Å (5), with an interpretation based on atomic models of the individual subunit structures (6-8), and are now available at atomic resolution (9). These structures have provided unparalleled insight into the mechanism of different steps of translation (1) as well as inhibition by antibiotics (10).Compared to the bacterial ribosome, the eukaryotic counterpart is more complicated, containing expansion segments (ES) and variable regions in the rRNA as well as many additional r proteins and r-protein extensions. Plant and fungal 80S ribosomes contain ∼5;500 nucleotides (nts) of rRNA and ∼80 r proteins, whereas bacterial 70S ribosomes comprise ∼4;500 nts and 54 r proteins. The additional elements present in eukaryotic ribosomes may reflect the increased complexity of translation regulation in eukaryotic cells, as evident for assembly, translation initiation, and development, as well as the phenomenon of localized translation (11-15).Early models for eukaryotic ribosomes were derived from electron micrographs of negative-stain or freeze-dried ribosomal particles (16) and localization of r proteins was attempted using immuno-EM and cross-linking approaches; see, for example, refs. 17-20. The first cryo-EM reconstruction of a eukaryotic 80S ribosome was reported for wheat germ (Triticum aestivum) at 38 Å (21). Initial core models for the yeast 80S ribosome were built at 15-Å resolution (22) by docking the rRNA s...

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and α-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation.